In the field of biotechnology, there is a strongly felt need to enhance the level of expression of genes introduced into the relative organisms. This level is often unsatisfactory, and represents a barrier to the industrial application of innovations in plant and animal biotechnology. There is a quantity of data to support the importance of the leader region in regulating the levels of gene expression, while there are various structural elements that characterize the regulation capacity thereof.
In this case, the untranslated leader sequence in 5′ (5′-UTR), as it is proposed in widely diffused vectors (e.g. pBI121 and derivatives, pCAMBIA and derivatives), has numerous defects which make it unsuitable to direct adequate levels of gene expression in genetically modified organisms. In particular, when yields are to be maximized (e.g. the use of plants as cellular factories for compounds useful to man), it is necessary to eliminate the production constraints exerted by the 5′-UTR sequence. To this end, the leader Ω (a sequence that exists naturally in tobacco mosaic virus, TMV) has been proposed in plants. However, this too has imperfections and redundancies that render it open to improvement.
It is known that the region poly(CAA) in the translational enhancer present in the leader Ω of TMV (Gallie and Walbot 1992 Nucleic Acids Res., 20, 4631-4638) significantly enhances the expression levels, that is, it has a positive effect on the translation levels of heterologous proteins in vitro and in vivo (Gallie et al. 1988a Nucleic Acids Res., 16, 883-893, Gallie et al. 1988b Nucleic Acids Res., 16, 8675-8694, Gallie 2002 Nucleic Acids Res., 30, 3401-3411). In the leader Ω, a poly(CAA) sequence is associated with 3 repeats of the sequence ACAATTAC (Gallie et al. 1988a), but detection studies have shown that the regulator element responsible for enhancing the expression levels may consist of a single copy of the sequence ACAATTAC in combination with the motif (CAA)n (Gallie and Walbot 1992).
It is also known that the transcription initiation site (Inr) of the CaMV 35S promoter (Guilley et al. 1982 Cell, 30, 763-773) favours an efficient capping of the mRNA.
Furthermore, it is known that many plant leaders (Bolle et al. 1996 Plant Mol. Biol. 32, 861-868) have a sequence rich in CT elements and that the CT-rich sequences can alter the transcription levels (Chen et al. 1996 J. Virol., 70, 8411-8421).
It is also known that sequences which have a length of more than 40 nucleotides promote the recognition of the first AUG as authentic initial translation codon (Kozak 1989 J. Cell. Biol., 108, 229-241). For example, it has been observed that the extension of the leader from 29 to 74 nt causes an increase in the translation level of mRNA in vitro (Kozak 1991, J. Biol. Chem., 266, 19867-19870) and in vivo (Gallie and Walbot 1992). Leader sequences with a greater content of A/T cause higher expression levels since the formation of segments of double strand mRNA, due to the folding of the molecule over itself, is discouraged. In fact, it is certain that such secondary structures have a depressing effect on the translation efficiency (Pelletier and Sonenberg 1985 Cell, 40, 515-526; Kozak 1986 Proc. Natl. Acad. Sci. USA, 83, 2850-2854). Moreover, it has been noticed that the introduction of portions of 5′-UTRs of viral origin into plant leaders can be reflected in an increase in the level of expression of reporter proteins (Dowson Day et al. 1993 Plant Mol. Biol., 23, 97-109).
Purpose of the present invention is therefore to obviate the shortcomings of the state of the art and to achieve a leader sequence that increases the expression levels of recombinant proteins in plants.
The Applicant has devised, tested and embodied the present invention to overcome the shortcomings of the state of the art and to obtain these and other purposes and advantages.